Copper(II) Complexes with 1-(Isoquinolin-3-yl)heteroalkyl-2-ones: Synthesis, Structure and Evaluation of Anticancer, Antimicrobial and Antioxidant Potential

Four copper(II) complexes, C1–4, derived from 1-(isoquinolin-3-yl)heteroalkyl-2-one ligands L1–4 were synthesized and characterized using an elemental analysis, IR spectroscopic data as well as single crystal X-ray diffraction data for complex C1. The stability of complexes C1–4 under conditions mimicking the physiological environment was estimated using UV-Vis spectrophotometry. The antiproliferative activity of both ligands L1–4 and copper(II) compounds C1–4 were evaluated using an MTT assay on four human cancer cell lines, A375 (melanoma), HepG2 (hepatoma), LS-180 (colon cancer) and T98G (glioblastoma), and a non-cancerous cell line, CCD-1059Sk (human normal skin fibroblasts). Complexes C1–4 showed greater potency against HepG2, LS180 and T98G cancer cell lines than etoposide (IC50 = 5.04–14.89 μg/mL vs. IC50 = 43.21–>100 μg/mL), while free ligands L1–4 remained inactive in all cell lines. The prominent copper(II) compound C2 appeared to be more selective towards cancer cells compared with normal cells than compounds C1, C3 and C4. The treatment of HepG2 and T98G cells with complex C2 resulted in sub-G1 and G2/M cell cycle arrest, respectively, which was accompanied by DNA degradation. Moreover, the non-cytotoxic doses of C2 synergistically enhanced the cytotoxic effects of chemotherapeutic drugs, including etoposide, 5-fluorouracil and temozolomide, in HepG2 and T98G cells. The antimicrobial activities of ligands L2–4 and their copper(II) complexes C2–4 were evaluated using different types of Gram-positive bacteria, Gram-negative bacteria and yeast species. No correlation was found between the results of the antiproliferative and antimicrobial experiments. The antioxidant activities of all compounds were determined using the DPPH and ABTS radical scavenging methods. Antiradical tests revealed that among the investigated compounds, copper(II) complex C4 possessed the strongest antioxidant properties. Finally, the ADME technique was used to determine the physicochemical and drug-likeness properties of the obtained complexes.


Introduction
Since metal-based compounds play remarkable roles as therapeutic and diagnostic agents, the search for novel metallopharmaceuticals represents an area of significant interest in the field of medicinal chemistry [1,2].The therapeutic potential of metal complexes has a long history; however, the discovery of the bacteriostatic and anticancer activity ruthenium(II) phenanthroline complexes by Francis Dwyer, followed by the discovery of the anticancer properties of cis-diaminodichloridoplatinum(II)-cisplatin by Barnet Rosenberg, was a milestone in the development of metal-containing drugs [1].Among clinically approved anticancer chemotherapeutics, platinum agents such as cisplatin and its derivatives, e.g., carboplatin oxaliplatin and picoplatin, are still the most prominent drugs used in the treatment of solid cancers [3].Nonetheless, despite the evident success of cisplatin and its analogues in medicine, their progress in clinical application is limited due to their well-known drawbacks, such as low solubility, severe side effects, including nephrotoxicity and neurotoxicity, and the intrinsic or acquired resistance of cancer cells to platinumcontaining drugs [4][5][6][7].Consequently, with the emergence of many biomedical applications of other transition metal complexes, including anticancer and antimicrobial agents, attention is shifting beyond platinum-based compounds [1,[8][9][10][11].The preclinical studies provide evidence that non-platinum agents have the potential to circumvent the problem of chemoresistance and the toxicity of platinum-based agents by exhibiting different specific modes of action, reduced undesirable effects and the ability to overcome drug-resistance mechanisms [10,12,13].
Antitumor agents based on endogenous metals such as cobalt, zinc, iron or copper were found to be less toxic compared to platinum analogues [14].Among them, copper(II)containing coordination compounds have attracted considerable interest due to the significant role of copper in cancer [15,16].The copper ion is involved in essential processes of cancer progression such as cell proliferation, angiogenesis or metastasis, and it provides various mechanisms of antitumor action [17][18][19].For example, the antiproliferative effect of copper(II) complexes results from the inhibition of the activities of enzymes, which play a pivotal role in cancer therapy, e.g., protein disulfide isomerase (PDI) [20], topoisomerases I and II [21][22][23], telomerase [24] or proteasome [25][26][27], as well as DNA intercalation [28][29][30] and DNA degradation [31,32].Moreover, the antitumor activity of copper compounds may also be the consequence of their ability to induce apoptosis [33,34] and non-apoptotic cell death-paraptosis [25,35]-reactive oxygen species (ROS) formation that triggers tumor cell death [36,37], and antiangiogenic properties [38].In turn, the copper(II) complex with disulfiram, a drug used in humans to treat alcoholism with great potential for the treatment of human cancers [39], was found to be capable of reversing the drug resistance of doxorubicin (ADM)-resistant acute leukemia cell lines by the induction of apoptosis [40].Additionally, it is well known that the copper coordination in organic compounds may lead to higher antitumor activity [23,41], selectivity [23] and reduced toxicity [42] compared to the free ligands.In this line, it is worth noting that the uptake of copper(II) complexes by cancer cells is higher than that by normal cells [38,43].In addition, copper(II) complexes may exhibit a different response to cancer cells than to non-cancerous cells [44].Overall, copper(II)-containing coordination compounds have emerged as a promising class of therapeutic anticancer agents with various mechanisms of action and the potential to overcome drug resistance [17,20,45].
It should be noted that the therapeutic potency of copper(II) complexes is not limited to anticancer activity.These compounds have also gained much interest due to their antiinflammatory and antioxidant properties [46][47][48], antiviral properties [49,50] or antibiofilm and antimicrobial [51,52] activities.The latter is a multi-faceted process, although the main mechanism of the bactericidal effect is the formation of ROS, causing irreversible damage to membranes [51].
Isoquinoline-containing compounds are of scientific interest due to their fluorescent properties and broad spectrum of biological activities including antihypertensive [53], antiinflammatory and analgesic [54] or antioxidant [55] activities, as well as their ability to act as antidepressant and antipsychotic [56] agents.Moreover, the importance of isoquinoline scaffold in drug design [57,58] has also led to the development of bioactive compounds with antimalarial [59], antifungal and antibacterial effects [60,61].In addition, isoquinoline derivatives constitute an important source of novel anticancer agents that may exert their biological activities through various mechanisms such as apoptosis, DNA fragmentation, the inhibition of tubulin polymerization, induced cell cycle arrest and the interruption of cell migration [62,63].On the other hand, the research on the biological activities of their metal complexes, especially copper(II)-coordination compounds, is not very extensive.
Our previous work indicated that the newly synthesized 1-(isoquinolin-3-yl)heteroalkyl-2-ones of type A (Figure 1) possess promising fluorescent properties [64].In the present work, as a continuation of a research program on the chemistry and biological activity of copper(II) complexes undertaken in our laboratory [64][65][66][67][68], we wish to report the results of studies on the reactions of the above-mentioned isoquinoline derivatives A with copper(II) chloride, an X-ray structure determination of the complexes obtained of type B (Figure 1), as well as the results of the evaluation of their anticancer, antimicrobial and antioxidant potential.Furthermore, to confirm the importance of copper coordination in organic ligands, the biological properties of the free ligands A were also evaluated.
Int. J. Mol.Sci.2024, 25, x FOR PEER REVIEW 3 of 29 to act as antidepressant and antipsychotic [56] agents.Moreover, the importance of isoquinoline scaffold in drug design [57,58] has also led to the development of bioactive compounds with antimalarial [59], antifungal and antibacterial effects [60,61].In addition, isoquinoline derivatives constitute an important source of novel anticancer agents that may exert their biological activities through various mechanisms such as apoptosis, DNA fragmentation, the inhibition of tubulin polymerization, induced cell cycle arrest and the interruption of cell migration [62,63].On the other hand, the research on the biological activities of their metal complexes, especially copper(II)-coordination compounds, is not very extensive.
Our previous work indicated that the newly synthesized 1-(isoquinolin-3-yl)heteroalkyl-2-ones of type A (Figure 1) possess promising fluorescent properties [64].In the present work, as a continuation of a research program on the chemistry and biological activity of copper(II) complexes undertaken in our laboratory [64][65][66][67][68], we wish to report the results of studies on the reactions of the above-mentioned isoquinoline derivatives A with copper(II) chloride, an X-ray structure determination of the complexes obtained of type B (Figure 1), as well as the results of the evaluation of their anticancer, antimicrobial and antioxidant potential.Furthermore, to confirm the importance of copper coordination in organic ligands, the biological properties of the free ligands A were also evaluated.

Synthesis of 1-(Isoquinolin-3-yl)heteroalkyl-2-one Ligands L1-4
Ligand 1-(isoquinolin-3-yl)azetidin-2-one (L1) was synthesized according to a previously described procedure involving copper-catalyzed Goldberg-Ullmann-type coupling of 3-bromoisoquinoline with azetidin-2-one [64].These reactions were carried out in anhydrous dioxane or n-butyl alcohol in the presence of a base, copper(I) iodide and N,Ndimethylethylenediamine (Scheme 1).Ligand 1-(isoquinolin-3-yl)azetidin-2-one (L1) was synthesized according to a previously described procedure involving copper-catalyzed Goldberg-Ullmann-type coupling of 3-bromoisoquinoline with azetidin-2-one [64].These reactions were carried out in anhydrous dioxane or n-butyl alcohol in the presence of a base, copper(I) iodide and N,N-dimethylethylenediamine (Scheme 1). to act as antidepressant and antipsychotic [56] agents.Moreover, the importance of isoquinoline scaffold in drug design [57,58] has also led to the development of bioactive compounds with antimalarial [59], antifungal and antibacterial effects [60,61].In addition, isoquinoline derivatives constitute an important source of novel anticancer agents that may exert their biological activities through various mechanisms such as apoptosis, DNA fragmentation, the inhibition of tubulin polymerization, induced cell cycle arrest and the interruption of cell migration [62,63].On the other hand, the research on the biological activities of their metal complexes, especially copper(II)-coordination compounds, is not very extensive.

Results and Discussion
Our previous work indicated that the newly synthesized 1-(isoquinolin-3-yl)heteroalkyl-2-ones of type A (Figure 1) possess promising fluorescent properties [64].In the present work, as a continuation of a research program on the chemistry and biological activity of copper(II) complexes undertaken in our laboratory [64][65][66][67][68], we wish to report the results of studies on the reactions of the above-mentioned isoquinoline derivatives A with copper(II) chloride, an X-ray structure determination of the complexes obtained of type B (Figure 1), as well as the results of the evaluation of their anticancer, antimicrobial and antioxidant potential.Furthermore, to confirm the importance of copper coordination in organic ligands, the biological properties of the free ligands A were also evaluated.

Synthesis of Copper(II) Complexes of 1-(Isoquinolin-3-yl)heteroalkyl-2-ones C1-4
Copper(II) complexes C1-4 were prepared through the reaction of copper(II) chloride dihydrate with previously described 1-(isoquinolin-3-yl)heteroalkyl-2-ones L1-4 [64].For the preparation of metal complexes that are stable under physiological conditions, reactions were carried out in dimethylformamide (DMF) containing 0.5% water and the dihydrate of copper(II) salt.The use of dimethylformamide as a solvent had a positive effect on the purity and further isolation of the desired copper(II) complexes.The formation of precipitate or crystals of green or brown copper(II) complexes was observed at room temperature over a period of 3 to 12 days.After the required time, the products-copper(II) complexes-were separated by filtration.
Firstly, we began by studying the equimolar ratio of ligands L1-4 and copper(II) salt in 99.5% dimethylformamide.It was found that for the copper(II) complexes C3 and C4, a stoichiometric ratio of the ligands L3 and L4 in terms of yields was the most important factor.In the case of copper(II) complexes C1 and C2, two molecules of a neutral bidentate ligand L1 or L2 can coordinate with the copper(II) ion.Thus, a 2-fold excess of ligands L1 Scheme 2. Synthesis of ligand L2 in the reaction of isoquinoline N-oxide with 2-chloroimidazoline and N-alkylated analogous L3 and L4.

Synthesis of Copper(II) Complexes of 1-(Isoquinolin-3-yl)heteroalkyl-2-ones C1-4
Copper(II) complexes C1-4 were prepared through the reaction of copper(II) chloride dihydrate with previously described 1-(isoquinolin-3-yl)heteroalkyl-2-ones L1-4 [64].For the preparation of metal complexes that are stable under physiological conditions, reactions were carried out in dimethylformamide (DMF) containing 0.5% water and the dihydrate of copper(II) salt.The use of dimethylformamide as a solvent had a positive effect on the purity and further isolation of the desired copper(II) complexes.The formation of precipitate or crystals of green or brown copper(II) complexes was observed at room temperature over a period of 3 to 12 days.After the required time, the products-copper(II) complexes-were separated by filtration.
Firstly, we began by studying the equimolar ratio of ligands L1-4 and copper(II) salt in 99.5% dimethylformamide.It was found that for the copper(II) complexes C3 and C4, a stoichiometric ratio of the ligands L3 and L4 in terms of yields was the most important factor.In the case of copper(II) complexes C1 and C2, two molecules of a neutral bidentate ligand L1 or L2 can coordinate with the copper(II) ion.Thus, a 2-fold excess of ligands L1 or L2 resulted in the creation of tetra-coordinate mononuclear copper(II) complexes C1 and C2, respectively.
Mononuclear complex C1 (L 2 CuCl 2 ) was obtained in the reaction of ligand L1 with copper(II) chloride dihydrate in a 1:1 molar ratio through the slow evaporation of the solvent at room temperature (Scheme 3).
or L2 resulted in the creation of tetra-coordinate mononuclear copper(II) complexes C1 and C2, respectively.
Mononuclear complex C1 (L2CuCl2) was obtained in the reaction of ligand L1 with copper(II) chloride dihydrate in a 1:1 molar ratio through the slow evaporation of the solvent at room temperature (Scheme 3).Scheme 3. Synthesis of copper(II) complex C1 in the reaction of ligand L1 with copper(II) chloride dihydrate in dimethylformamide.
The coordination compounds C2, C3 and C4 were obtained in acceptable yields with analogous reaction conditions and copper(II) salt stoichiometries (Scheme 4).It should be emphasized that the ligand unsubstituted at the nitrogen atom in position 3 of the imidazolidin-2-one; derivative L2 formed a neutral mononuclear chelate C2 (L2CuCl2), while the N 3 -substituted ligands L3 and L4 allowed the preparation of brown-green bidentate N,O-chelates C3 and C4 with the structure LCuCl2.Summing up, the efficiency of the complexation reactions, calculated as the ratio of the achieved yield to the theoretical yield, was approximately twice higher (61-68%) in the case of the bidentate N,O-chelates C3 and C4 (LCuCl2) than the mononuclear complexes C1 and C2 with the L2CuCl2 structure (27-33%).The coordination compounds C2, C3 and C4 were obtained in acceptable yields with analogous reaction conditions and copper(II) salt stoichiometries (Scheme 4).It should be emphasized that the ligand unsubstituted at the nitrogen atom in position 3 of the imidazolidin-2-one; derivative L2 formed a neutral mononuclear chelate C2 (L 2 CuCl 2 ), while the N 3 -substituted ligands L3 and L4 allowed the preparation of brown-green bidentate N,O-chelates C3 and C4 with the structure LCuCl 2 .
or L2 resulted in the creation of tetra-coordinate mononuclear copper(II) complex and C2, respectively.
Mononuclear complex C1 (L2CuCl2) was obtained in the reaction of ligand L copper(II) chloride dihydrate in a 1:1 molar ratio through the slow evaporation of t vent at room temperature (Scheme 3).Summing up, the efficiency of the complexation reactions, calculated as the ratio achieved yield to the theoretical yield, was approximately twice higher (61-68%) case of the bidentate N,O-chelates C3 and C4 (LCuCl2) than the mononuclear com C1 and C2 with the L2CuCl2 structure (27-33%).Summing up, the efficiency of the complexation reactions, calculated as the ratio of the achieved yield to the theoretical yield, was approximately twice higher (61-68%) in the case of the bidentate N,O-chelates C3 and C4 (LCuCl 2 ) than the mononuclear complexes C1 and C2 with the L 2 CuCl 2 structure (27-33%).

Structural Analysis of Copper(II) Complexes C1-4
The structures of copper(II) complexes C1-4 were confirmed using an elemental analysis and infrared spectroscopic data.Moreover, the crystal structure of copper(II) complex C1 was determined using X-ray crystallography.It should be mentioned that the presence of an unpaired electron attributed to the copper(II) ion in complexes C1-4 results in their paramagnetic properties; thus, the nuclear magnetic resonance (NMR) spectra of compounds C1-4 cannot be recorded.

Infrared Spectra
In the infrared spectra of copper(II) complexes C1, C2, C3 and C4 stretching vibrations of the carbonyl group (C=O) were observed in the range of 1639 to 1751 cm −1 .It should be noted that the IR spectra of the copper(II) complexes showed the characteristic shifts of functional group absorptions, confirming their involvement in the chelation of a metal ion.Hence, in the case of synthesized complexes, shifted stretching bands were observed for the C=O group of the lactam (C1) or cyclic urea ring (C2, C3 and C4), and the C=N moiety of the isoquinoline ring (Figures S1-S8, Supplementary Materials).
The structures of copper(II) complexes C1-4 were confirmed using an elemental anal-ysis and infrared spectroscopic data.Moreover, the crystal structure of copper(II) complex C1 was determined using X-ray crystallography.It should be mentioned that the presence of an unpaired electron attributed to the copper(II) ion in complexes C1-4 results in their paramagnetic properties; thus, the nuclear magnetic resonance (NMR) spectra of compounds C1-4 cannot be recorded.

Infrared Spectra
In the infrared spectra of copper(II) complexes C1, C2, C3 and C4 stretching vibrations of the carbonyl group (C=O) were observed in the range of 1639 to 1751 cm −1 .It should be noted that the IR spectra of the copper(II) complexes showed the characteristic shifts of functional group absorptions, confirming their involvement in the chelation of a metal ion.Hence, in the case of synthesized complexes, shifted stretching bands were observed for the C=O group of the lactam (C1) or cyclic urea ring (C2, C3 and C4), and the C=N moiety of the isoquinoline ring (Figures S1-S8, Supplementary Materials).

X-ray Crystallographic Studies
The crystal data, data collection and structure refinement details are summarized in Table 1.

X-ray Crystallographic Studies
The crystal data, data collection and structure refinement details are summarized in Table 1.In the crystal structure of complex C1, the organic ligand L1 is coordinated in the bidentate manner via the oxygen atom of the carbonyl group and the nitrogen atom of the isoquinoline ring, forming a six-membered chelate cycle (Figure 3).In the crystal structure of complex C1, the organic ligand L1 is coordinated in the bidentate manner via the oxygen atom of the carbonyl group and the nitrogen atom of the isoquinoline ring, forming a six-membered chelate cycle (Figure 3).There are no strong hydrogen bonds in the C1 structure.There are hydrogen bonds of the C-H…Cl(O) type.The C-H…O bond stabilizes the coordination fragment (Table 2).However, the C-H…Cl hydrogen bond stabilizes the packing.

Molecular Modeling Studies of Ligands L1 and L2
Our previous X-ray studies indicated that in the crystal state, the compound L2 adopts the E conformation, which is probably stabilized by intramolecular C-H• • • O (2.26 Å).The molecules of ligand L2 are connected by pairs of N-H• • • O hydrogen bonds (1.99 Å) between two imidazolidin-2-one ring fragments with the formation of centrosymmetric dimers [64].The many possible rotamers of the representative ligand-1-(isoquinolin-3-yl)imidazolidin-2-one (L2)-can be generated from the rotation around the bond axis C3 (isoquinoline) -N1 (imidazolin-2-one) (Figure 5).Table 2. Hydrogen bond geometry for copper(II) complex C1.Some additional geometrical details can be found in the Supplementary Ma (Tables S1-S3).

Molecular Modeling Studies of Ligands L1 and L2
Our previous X-ray studies indicated that in the crystal state, the compou adopts the E conformation, which is probably stabilized by intramolecular C-H⋯O Å).The molecules of ligand L2 are connected by pairs of N-H⋯O hydrogen bond Å) between two imidazolidin-2-one ring fragments with the formation of centrosy ric dimers [64].The many possible rotamers of the representative ligand-1-(isoqu 3-yl)imidazolidin-2-one (L2)-can be generated from the rotation around the bon C3(isoquinoline)-N1′(imidazolin-2-one) (Figure 5).In this study, the X-ray diffraction analysis results obtained for copper(II) complex C1 showed that two molecules of ligand L1 exist in the conformation E. For this reason, we decided to perform quantum chemical calculations to gain a better understanding of the structure of the ligands L1-4.We assumed that the formation of the copper(II) complexes of ligands L1-4 requires a rotation of their conformation from E to Z.
The structures of the selected two ligands L1 and L2 were optimized in a polar solvent (DMF) by using the Spartan program suite (Spartan version 14 V 1.1.4.).The possible conformers of compounds L1 and L2 were calculated at the B3LYP/6.31G**level of theory [71][72][73].It should be mentioned that B3LYP-the so-called 'hybrid functional'-is one of the most popular DFT functionals used for the prediction of the physicochemical properties of molecules in in silico drug design [74].
In the case of ligand L1, the structure with a torsion angle (N 2 -C 3 -N 1 -C 2 , Φ = 180 • ) was proven to be the lowest energy rotameric form in the DMF solution (E conformation of molecule) (Figure 6).The energy difference between the E conformation and its rotamer in the Z conformation (N 2 -C 3 -N 1 -C 2 , Φ = 0) was calculated to be ∆E = 8.146 kcal/mol.Based on this, it may be concluded that the barrier to the C 3 -N 1 bond rotation is low, and it is easy to overcome the energy barrier under normal conditions.This suggests that rotamers having different torsion angles may exist together in the solution at room temperature.In the Z conformation of 1-(isoquinolin-3-yl)azetidin-2-one (L1), the position of the nitrogen and oxygen atoms of the two heterocyclic rings is favored for the chelation of copper(II) ions.
In the case of ligand L1 in its E conformation with torsion angle Φ = 0, the highest occupied molecular orbital (E HOMO = −5.67 eV) is confined to carbon atoms C 3 -C 8 of the isoquinoline ring, and nitrogen (N 1 ) and carbon (C 2 ) atoms of the azetydin-2-one system, while the frontier orbital LUMO (E LUMO = −1.52 eV) is located mostly on the entire isoquinoline scaffold (Figure 7).The calculated HOMO-LUMO energy gap for ligand L1 in conformation with the torsion angle Φ = 0 (E g = 4.15 eV) is lower than the energy gap obtained for its Φ = 180 rotamer (E g = 4.27 eV).This may suggest the higher reactivity of conformer L1 with the torsion angle Φ = 0 towards bonding with transient metals such as copper.
in the Z conformation (N2-C3-N1′-C2′, Φ = 0) was calculated to be ΔE = 8.146 kcal/mol.Based on this, it may be concluded that the barrier to the C3-N1′ bond rotation is low, and it is easy to overcome the energy barrier under normal conditions.This suggests that rotamers having different torsion angles may exist together in the solution at room temperature.In the Z conformation of 1-(isoquinolin-3-yl)azetidin-2-one (L1), the position of the nitrogen and oxygen atoms of the two heterocyclic rings is favored for the chelation of copper(II) ions.In the case of ligand L1 in its E conformation with torsion angle Φ = 0, the highest occupied molecular orbital (EHOMO = −5.67 eV) is confined to carbon atoms C3-C8 of the isoquinoline ring, and nitrogen (N1′) and carbon (C2′) atoms of the azetydin-2-one system, while the frontier orbital LUMO (ELUMO = −1.52 eV) is located mostly on the entire isoquinoline scaffold (Figure 7).The calculated HOMO-LUMO energy gap for ligand L1 in conformation with the torsion angle Φ = 0 (Eg = 4.15 eV) is lower than the energy gap obtained for its Φ = 180 rotamer (Eg = 4.27 eV).This may suggest the higher reactivity of conformer L1 with the torsion angle Φ = 0 towards bonding with transient metals such as copper.Similarly, the in silico data for ligand L2 revealed that conformer with torsion N2-C3-N1′-C2′ at about Φ = 172 was calculated to be slightly lower in energy −441,256.812142kcal/mol) than the conformer with torsion angle Φ = 0 (E = −441,250.kcal/mol) (Figure 8).The energy difference between these two rotamers was estima be ΔE = 5.845 kcal/mol.Moreover, based on calculated dipole moments, the confo with torsion angle Φ = 0 (µ = 6.60 debye)-favored for the binding of copper(II) i would be predicted to prevail over the second one (Φ = 172, µ = 3.23 debye) in a solvent such as dimethylformamide.Similarly, the in silico data for ligand L2 revealed that conformer with torsion angle N 2 -C 3 -N 1 -C 2 at about Φ = 172 was calculated to be slightly lower in energy (E = −441,256.812142kcal/mol) than the conformer with torsion angle Φ = 0 (E = −441,250.96698kcal/mol) (Figure 8).The energy difference between these two rotamers was estimated to be ∆E = 5.845 kcal/mol.Moreover, based on calculated dipole moments, the conformer with torsion angle Φ = 0 (µ = 6.60 debye)-favored for the binding of copper(II) ions-would be predicted to prevail over the second one (Φ = 172, µ = 3.23 debye) in a polar solvent such as dimethylformamide.
Based on this, it may be concluded that for ligands L1 and L2, the barrier to the C 3 -N 1 bond rotation is low, and it is easy to overcome the energy barrier under normal conditions (at room temperature) and in polar solvents (dimethylformamide).This suggests that rotamers of ligands L1-4 having different torsion angles may exist in the solution at room temperature.In their Z conformations, the position of the atoms having donating properties is favored for the binding of copper(II) ions.Therefore, forming a six-membered chelate ring requires energy, which can be compensated for by creating novel bonds involving copper and nitrogen or oxygen atoms: Cu 2+ ---N=C and Cu 2+ ---O=C.
−441,256.812142kcal/mol) than the conformer with torsion angle Φ = 0 (E = −441,250.96698kcal/mol) (Figure 8).The energy difference between these two rotamers was estimated to be ΔE = 5.845 kcal/mol.Moreover, based on calculated dipole moments, the conformer with torsion angle Φ = 0 (µ = 6.60 debye)-favored for the binding of copper(II) ionswould be predicted to prevail over the second one (Φ = 172, µ = 3.23 debye) in a polar solvent such as dimethylformamide.Based on this, it may be concluded that for ligands L1 and L2, the barrier to the C3-N1′ bond rotation is low, and it is easy to overcome the energy barrier under normal conditions (at room temperature) and in polar solvents (dimethylformamide).This suggests that rotamers of ligands L1-4 having different torsion angles may exist in the solution at room temperature.In their Z conformations, the position of the atoms having donating properties is favored for the binding of copper(II) ions.Therefore, forming a six-membered chelate ring requires energy, which can be compensated for by creating novel bonds involving copper and nitrogen or oxygen atoms: Cu 2+ ---N=C and Cu 2+ ---O=C.

Stability Studies of Copper(II) Complexes C1-4 in Aqueous Buffer
The stability testing of the synthesized compounds must validate the biological results by ensuring that the compound remains biologically active over time.It should be emphasized that the limited stability of drug candidates in physiological pH ranges prevents their use in vivo.In the case of metal complexes, this is a crucial aspect due to the fact that they may dissociate under physiological conditions, releasing the metal ions and the free ligands.

Stability Studies of Copper(II) Complexes C1-4 in Aqueous Buffer
The stability testing of the synthesized compounds must validate the biological results by ensuring that the compound remains biologically active over time.It should be emphasized that the limited stability of drug candidates in physiological pH ranges prevents their use in vivo.In the case of metal complexes, this is a crucial aspect due to the fact that they may dissociate under physiological conditions, releasing the metal ions and the free ligands.
To address this question and confirm the validity of the biological results, we performed UV-vis stability measurements of copper(II) complexes C1-4 under conditions that mimic the physiological environment (phosphate buffered aqueous solution, pH = 7.4, 37 • C).An increase in absorbance during the measurements may indicate the release of free ligands, whereas a decrease in absorbance shows the precipitation of the complex from the buffer solution.
Firstly, copper(II) complexes were dissolved in 99.5% DMF at a concentration of 4 mM.These solutions were diluted into a 100 mM potassium phosphate buffer with pH 7.4 to a final concentration of 40 µM of copper(II) complex in a quartz cuvette at a temperature of 37 • C. To identify very small changes in the UV-vis spectra, difference spectra between λ = 250-600 nm were recorded every 10 min with a diode array photometer over the course of 3 h at 37 • C. The difference spectra were obtained by subtracting the spectrum at time = 0 from each of the following recorded spectra between λ = 250 and 600 nm.
The tested copper(II) complexes C1-4 did not show noticeable changes in their timedependent difference spectra over 3 h of incubation in the phosphate-buffered aqueous solution (pH 7.4, 37 • C).It was observed that the intensity did not change during the experiments.It is also worth noting the lack of isosbestic points in the range of 250 and 600 nm.The presence of isosbestic points indicates a possibility of reaction in the buffered solution, for example, ligand exchange.Thus, the complexes C1-4 appear stable under biologically similar conditions with no apparent loss of the Cu(II) from the ligand.
Figure 9 presents the time-dependent UV-Vis spectra of the representative copper(II) complex C1.The UV-vis difference spectra of copper(II) complexes C2, C3 and C4 are shown in the supporting information (Figures S9-S12, Supplementary Materials).
periments.It is also worth noting the lack of isosbestic points in the range of 250 and 600 nm.The presence of isosbestic points indicates a possibility of reaction in the buffered solution, for example, ligand exchange.Thus, the complexes C1-4 appear stable under biologically similar conditions with no apparent loss of the Cu(II) from the ligand.
Figure 9 presents the time-dependent UV-Vis spectra of the representative copper(II) complex C1.The UV-vis difference spectra of copper(II) complexes C2, C3 and C4 are shown in the supporting information (Figures S9-S12, Supplementary Materials).

In Vitro Cytotoxic Activity
The in vitro cytotoxic activities of free ligands L1-4 and the corresponding copper(II) complexes C1-4 were evaluated against four human cancer cell lines, namely melanoma A375, hepatoma HepG2, colon cancer LS-180 and glioblastoma T98G.To establish the selectivity towards cancer cell lines, the investigated compounds were also tested on a noncancerous human skin fibroblast cell line-CCD-1059-Sk.
From the results presented in Table 3, it is apparent that free ligands L1, L2, L3 and L4 were inactive in all cell lines up to 200 µg/mL, while their copper(II) complexes C1, C2, C3 and C4 exhibited remarkable growth inhibitory potency towards cancer cell lines (IC50

Biological Evaluation 2.4.1. In Vitro Cytotoxic Activity
The in vitro cytotoxic activities of free ligands L1-4 and the corresponding copper(II) complexes C1-4 were evaluated against four human cancer cell lines, namely melanoma A375, hepatoma HepG2, colon cancer LS-180 and glioblastoma T98G.To establish the selectivity towards cancer cell lines, the investigated compounds were also tested on a non-cancerous human skin fibroblast cell line-CCD-1059-Sk.
From the results presented in Table 3, it is apparent that free ligands L1, L2, L3 and L4 were inactive in all cell lines up to 200 µg/mL, while their copper(II) complexes C1, C2, C3 and C4 exhibited remarkable growth inhibitory potency towards cancer cell lines (IC 50 values ranging from 5.04 µg/mL to 37.97 µg/mL) compared with the widely used anticancer agent-etoposide (IC 50 values between 10.20 and >100 µg/mL).It should be noted that copper(II) compounds C1-4 showed several times greater effectiveness against cancer cell lines than the reference drug (IC 50 = 5.04-14.89µg/mL vs. IC 50 = 43.21->100µg/mL); the exception to this was the melanoma A375 cell line, which was the least susceptible to the effect of the tested complexes (IC 50 = 22.78-37.97µg/mL vs. IC 50 = 10.20 µg/mL).Among the tested complexes, compound C2 bearing imidazolidin-2-one moiety was found to be the most potent on the HepG2 and T98G cancer cell lines (Table 3).Its analogues bearing the methyl or ethyl substituent at the R position of the imidazolidin-2-one scaffold (C3: R = CH 3 ; C4: R = C 2 H 5 ) displayed slightly weaker antiproliferative activities (IC 50 = 5.04-6.97µg/mL vs. IC 50 = 6.72-12.00µg/mL).Furthermore, complex C1 with an azetidin-2-one functionality showed a comparable level of ability to inhibit the growth of the HepG2 and T98G cancer cell lines with the complexes C3 and C4 featuring the imidazolidin-2-one moiety (IC 50 = 8.25-14.89µg/mL vs. IC 50 = 6.72-12.00µg/mL), although compound C1 turned out to be the least active in all cancer cell lines (IC 50 = 14.89-37.97µg/mL) (Table 3).
Summing up, the data presented here confirmed the hypothesis that the introduction of a metal ion into organic ligands may have a beneficial effect on the anticancer potential [11,23,38,43,75].

Cell Cycle Analysis
Since copper(II) complex C2 most effectively inhibited the growth of HepG2 and T98G cancer cells, its effect on cell cycle progression was examined by using the cytometry method (Figure 10).Interestingly, this compound revealed a distinct effect on the cell cycle progression of HepG2 and T98G cells.The growth inhibition of HepG2 cells was associated with cell cycle arrest in the sub-G1 phase, showing low-molecular-weight fragments of DNA as the evidence of apoptosis (Figure 10A).When analyzing the DNA content in the sub-G1 phase, a significant increase (p < 0.0001) from 10% in the control cells to 29% in the C2(CX)-treated HepG2 cells was observed.The identification of the occurrence of apoptosis on the basis of the elevated number of cells in the sub-G1 phase relies on the principle that degraded DNA fragments (i.e., early signs of apoptosis) are released from cells, which results in the increased number of cells possessing a reduced DNA content.Consecutively, the cytotoxic effect of C2 (CX) on the T98G cells resulted from cell cycle arrest in the G2/M phase, which indicates considerable DNA damages that are unable to be repaired before mitosis (Figure 10B).

Interaction of Copper(II) Complex C2 with Clinically Used Anticancer Agents
Possible interactions between anticancer drugs should be an important consideration among patients undergoing antineoplastic therapy since they are exposed to several types of treatments, each including a number of drugs.As most of them have a narrow therapeutic index, it is important to examine the possible new strategies that can increase the clinical outcomes by using lower doses of the currently available anticancer drugs.Such cotreatments can also be an effective strategy for overcoming resistance in cancer therapy.In our studies, copper(II) complex C2, which exhibited the most potent effect against HepG2 and T98G cancer cells, was selected to examine the possible synergism with clinically used anticancer agents.The combinations of C2 (CX) and etoposide (ETO), cisplatin (CIS) and 5-fluorouracil (5-FU) were evaluated against both of the cell lines (Figures 11 and 12).Additionally, the combination of C2 (CX) and temozolomide (TMZ)-a chemotherapeutic agent being used as a first-line treatment for glioblastoma-was tested on the T98G glioblastoma cell line (Figure 11).
the C2(CX)-treated HepG2 cells was observed.The identification of the occurrence of apoptosis on the basis of the elevated number of cells in the sub-G1 phase relies on the principle that degraded DNA fragments (i.e., early signs of apoptosis) are released from cells, which results in the increased number of cells possessing a reduced DNA content.Consecutively, the cytotoxic effect of C2 (CX) on the T98G cells resulted from cell cycle arrest in the G2/M phase, which indicates considerable DNA damages that are unable to be repaired before mitosis (Figure 10B).

Interaction of Copper(II) Complex C2 with Clinically Used Anticancer Agents
Possible interactions between anticancer drugs should be an important consideration among patients undergoing antineoplastic therapy since they are exposed to several types of treatments, each including a number of drugs.As most of them have a narrow therapeutic index, it is important to examine the possible new strategies that can increase the clinical outcomes by using lower doses of the currently available anticancer drugs.Such co-treatments can also be an effective strategy for overcoming resistance in cancer therapy.In our studies, copper(II) complex C2, which exhibited the most potent effect against HepG2 and T98G cancer cells, was selected to examine the possible synergism with clinically used anticancer agents.The combinations of C2 (CX) and etoposide (ETO), cisplatin (CIS) and 5-fluorouracil (5-FU) were evaluated against both of the cell lines (Figures 11 and  The investigated drugs represent different mechanisms of anticancer activity, ing human DNA topoisomerase IIα inhibitors (ETO) [76], alkylating agents (CIS, [77,78], thymidylate synthase inhibitors and antimetabolites (5-FU) [79].Compou The investigated drugs represent different mechanisms of anticancer activity, including human DNA topoisomerase IIα inhibitors (ETO) [76], alkylating agents (CIS, TMZ) [77,78], thymidylate synthase inhibitors and antimetabolites (5-FU) [79].Compound C2 (CX), at the concentration that did not inhibit the growth of cancer cells (i.e., 0.25 µg/mL), statistically significantly improved the cytotoxic effects of ETO, 5-FU and TMZ against HepG2 and T98G cells (Figures 11 and 12).To better understand the mechanism of synergism between C2 and the clinically used drugs, it is important to gain insight into the mechanism of action of C2 alone.Usually, beneficial drug-drug interactions (synergism) can be expected when the components of the drug mixture act via different mechanisms.The investigated drugs represent different mechanisms of anticancer activity, including human DNA topoisomerase IIα inhibitors (ETO) [76], alkylating agents (CIS, TMZ) [77,78], thymidylate synthase inhibitors and antimetabolites (5-FU) [79].Compound C2 (CX), at the concentration that did not inhibit the growth of cancer cells (i.e., 0.25 µg/mL), statistically significantly improved the cytotoxic effects of ETO, 5-FU and TMZ against HepG2 and T98G cells (Figures 11 and 12).
To better understand the mechanism of synergism between C2 and the clinically used drugs, it is important to gain insight into the mechanism of action of C2 alone.Usually, beneficial drug-drug interactions (synergism) can be expected when the components of the drug mixture act via different mechanisms.
In this context, it should be mentioned that metal complexes with isoquinoline derivatives may exert anticancer properties through S-phase cell cycle arrest by the up-regulation of p53, p27 and p21 proteins and the down-regulation of cyclin A and cyclin E [80], mitochondrial (intrinsic) pathway-dependent apoptosis [81,82], caspase-3 activation triggering apoptosis [83], the inhibition of telomerase activity [75] as well as DNA intercalation [81,83].
In our studies, the cytotoxic activity of copper(II) complexes C2 against the tested cancer cell lines appears to be the result of cell cycle arrest in the sub-G1 phase (HepG2 cells) or G1/M phase (T98G cells) associated with DNA damage.Nevertheless, more work is needed to clarify the molecular mechanism of action of compound C2 leading to apoptosis.
The tested compounds did not show antibacterial activity against Gram-positive nor Gram-negative bacteria (MIC > 1000 mg/L,).As revealed by the data in Table 4, they displayed mild or no bioactivity towards the yeasts that were tested, except for ligand L3, which demonstrated moderate anti-Candida activity (MIC = 125-250 mg/L).Considering the promising anticancer potential of the tested complexes, it is notable that no antimicrobial activity is a beneficial property of these complexes as they do not cause harm to human microbiota during treatment.The gut microbiota plays a significant role in maintaining normal gut physiology and body health.It includes protection from pathogens by colonizing mucosal surfaces, producing different antimicrobial and enhancing the immune system, playing a significant role in digestion and metabolism, as well as influencing brain-gut communication and thus impacting the mental and neurological functions of the host [84].

Determination of Free Radical Scavenging Capacity
The disturbed redox balance between reactive oxygen species (ROS) and the antioxidant system is a critical factor in cancer development.One of the strategies for reducing tumors is targeting the redox metabolism by increasing the antioxidant capacity of cancer cells.In this way, antioxidants are being studied to develop more effective anticancer therapy [85].Furthermore, it has been reported that some oxidants may act as the inducers of DNA damage response, which may result in cell death [86,87].
On the other hand, in the ABTS assay, all tested compounds displayed antioxidant capacity with IC 50 values in the range from 72.5 µg/mL to 183.21 µg/mL (Table 5).The free ligands L2 and L3 demonstrated slightly higher ABTS quenching ability when compared to their complexes C2 and C3 (IC 50 = 82.08 and 96.76 µg/mL vs. IC 50 = 107.14 and 106.19 µg/mL, respectively), while ligands L1 and L4 proved to be less potent than the corresponding complexes C1 and C4 (IC 50 = 183.21and 108.59 µg/mL vs. IC 50 = 112.67 and 72.7 µg/mL, respectively).As in the DPPH assay, the most promising antiradical properties for ABST radical scavenging ability were presented by complex C4 (IC 50 = 72.7 µg/mL).
It could be concluded that in the DPPH assay, the coordination of ligands to the copper(II) metal center appears to be beneficial for antiradical potency as was previously reported [88][89][90].In general, no similar correlation was found for complexes C1-4 compared with their ligands L1-4 in the ABTS analysis.Nevertheless, it should be pointed out that the highest scavenging activity on both the DPPH and ABTS radicals was exhibited by Cu(II) complex C4.This observation suggests that the presence of the electron-donating ethyl group at the N-3 position of the imidazolidin-2-one moiety (R = C 2 H 5 ) facilitates antioxidant activity in the complex C4 by increasing the electron density at the central ion, leading to improved radical scavenging ability in the molecule [91].However, it was not possible to derive a correlation between antioxidant and antiproliferative activities with the only exception of Cu(II) complex C4, which demonstrated remarkable activity on the cancer cell lines, especially HepG2, LS180 and T98G (IC 50 = 5.92-9.27µg/mL), and the strongest antioxidant properties within the tested group (IC 50 = 26.46µg/mL in DDPH and 72.7 µg/mL in ABTS).On the contrary, the most potent complex against cancer cells, copper(II) complex C2 (IC 50 = 5.04-6.97µg/mL), exhibited less antiradical potency (IC 50 = 401.52µg/mL in DDPH, and 107.13 µg/mL in ABTS).
It is worth noting that due to the redox activity of the copper(II) complexes, some of the previously reported copper(II) compounds combine both antioxidant and pro-oxidant modes of action, inducing apoptosis in tumor cells [87].However, regarding the results of our studies, further work will be needed to clarify this.

In Silico Physicochemical, Pharmacokinetic and Drug-Likeness Predictions
The basic features of a drug molecule that determine whether it can be absorbed and transported inside the body include its solubility, lipophilicity, charge and size.Lipinski's rules dictate that undissociated substances with molecular weights below 500 Da, and a lipophilicity level in the range of 1-3 will have the best absorption rate.
The estimation of drug likeliness and the prediction of the physicochemical and pharmacokinetic properties-ADME (absorption, distribution, metabolism and excretion)-of copper(II) complexes C1, C2, C3 and C4 were carried out using the free available web tool SwissADME [92].The prediction of the principal properties of the molecules was carried out by using Lipinski's filter, which confirmed the drug likeness of the synthesized copper(II) complexes.The results of the calculated basic parameters of Cu(II) complexes C1, C2, C3 and C4 are presented in Table 6 and Figures 13 and 14 (for more details, see Table S4 in the Supplementary Materials).The topological polar surface area (TPSA) of a molecule can be defined as the sum of the polar atoms, namely oxygen and nitrogen, as well as their hydrogen atoms attached.A heightened TPSA rate (>140 Å 2 ) may be attributed to poor membrane permeability and blood-brain barrier accessibility.Thus, it can be said that a TPSA is a metric for describing the ability of compounds to permeate living cells [93].
As can be seen from the data in Table 6, copper(II) complexes C1-4 are characterized by reasonable polarity.Their TPSA values are in the range of 33.20-66.40Å 2 , except for compound C4, which has an estimated value equaling 90.46 Å 2 .All complexes possess a suitable lipophilicity estimated as a partition coefficient between n-octanol and water, with consensus logP (ClogP) values ranging from 1.87 to 3.04.Moreover, three copper(II) complexes, C1, C3 and C4, are expected to be moderately soluble in water.Lipinski's rule of five indicates that the lead compound should not contain more than 5 hydrogen bond donors (HBD), while hydrogen bond acceptors (HBAs) should not exceed 10.The calculated copper(II) complexes C1-4 exhibited two or four HBAs, and the aforementioned standard was congregated.According to the Veber's rule (rotatable bonds must be equivalent to 10 and PSA must be lower than 140 Å), the complexes are also expected to possess high oral bioavailability.
Moreover, according to Table 6, the bioavailable radar charts in Figure 13 and the "BOILED-egg" plot in Figure 14, the investigated copper(II) complexes C1, C2, C3 and C4 are predicted to possess high gastrointestinal tract (GI) absorption and blood-brain barrier BBB permeant.In this regard, all of the tested molecules show the same bioavailability score of 0.55, which suggests desirable pharmacokinetic properties.
In light of Lipinski's "rule of five", copper(II) complexes C1 and C2 slightly exceed the molecular weight and violate this criterion, while copper(II) complexes C3 and C4 meet all of the criteria as one of the key drug-likeness characteristics.Furthermore, according to Table 6, the ADME properties of copper(II) complexes are favorable and indicate that designed compounds may be considered drug-likeness molecules.

General Information
Melting points were determined with a Boëtius apparatus and are uncorrected.The infrared spectra were obtained on KBr pellets using a Nicolet 380 FT-IR 1600 spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA).The elemental analyses for C, H and N were within 0.4% of the theoretical values.Thin layer chromatography (TLC) of ligands L1, L2, L3 and L4 was performed on silica gel precoated 60 F254 Merck plates (Merck KGaA, Darmstadt, Germany) using the following eluents: chloroform and ethyl acetate (8:2, v/v), chloroform and methanol (99:1, v/v) or chloroform, ethyl acetate and methanol (8:1.5:0.5, v/v/v).The developed chromatograms were viewed under UV light at 254 nm.
The mass spectrum of ligand L4 was recorded on a Shimadzu LCMS-2010 EV (Tokyo, Japan) spectrometer equipped with an electrospray source, and the ESI-MS spectrum was registered in positive ion mode.
The difference spectra of copper complexes C1, C2, C3 and C4 from λ = 250 nm to 600 nm were recorded at 37 • C with a UV-VIS spectral photometer, Specord S600 (Analytik Jena, Jena, Germany), over the course of 3 h, with spectra being recorded every 10 min.Copper(II) complexes were dissolved in dimethylformamide (DMF) (Sigma-Aldrich Chemie GmbH, Steinheim, Germany).Freshly prepared solution of copper(II) complex at concentration 4 mM (25 µL) was added to 2.475 mL of 100 mM phosphate-buffered solution (pH 7.4), giving the complex a final concentration of 40 µM.Baseline correction was carried out by subtracting the mean absorption between λ = 250 and 600 nm from each spectrum.
The ligands L1 and L3, and the new ligand L4, were prepared as previously reported [64].Ligand L2 was synthesized using a modified method [69].

Aqueous Stability Studies
The stability of copper(II) complexes C1, C2, C3 and C4 was determined in 100 mM phosphate-buffered solution (pH 7.4) at 37 • C by using a Specord S600 (Analytic Jena, Jena, Germany) UV-vis diode-array photometer.Difference spectra were recorded every 10 min to help identify very small changes in the UV-vis spectra over 3 h incubations.formed in order to examine the possible interactions between compound C2 (CX) and ETO, CIS, 5-FU and TMZ.

In Vitro Antimicrobial Activity
Antibacterial and antifungal activities of the free ligands L2, L3 and L4 and the corresponding copper(II) complexes C2, C3 and C4 were screened using the two-fold microdilution broth method.Minimal inhibitory concentrations (MICs) of tested compounds for the panel of reference Gram-positive bacteria, including Staphylococcus aureus ATCC 25923, Staphylococcus aureus ATCC BAA-1707, Staphylococcus epidermidis ATCC 12228, Micrococcus luteus ATCC 10240 and Bacillus cereus ATCC 10876; Gram-negative bacteria, including Salmonella Typhimurium ATCC 14028, Escherichia coli ATCC 25922, Proteus mirabilis ATCC 12453, Klebsiella pneumoniae ATCC 13883 and Pseudomonas aeruginosa ATCC 9027; and yeasts, including Candida albicans ATCC 102231, Candida parapsilosis ATCC 22019 and Candida glabrata ATCC 90030 were determined.The procedure has been described in detail before [102].Briefly, the solutions of tested compounds dissolved in dimethylosulfoxide (DMSO) were suspended in Mueller-Hinton broth for bacteria or Mueller-Hinton broth with 2% glucose for fungi.Then, the series of two-fold dilutions were carried out in the sterile Nunc™ MicroWell™ 96-Well Microplates (ThermoFisher Scientific Inc.), obtaining concentrations from 1000 to 7.8 mg/L in the appropriate medium.Simultaneously, the inocula of 24 h cultures of microorganisms in sterile physiological saline (0.5 McFarland standard density) were prepared and added to each well, obtaining final density of 5 × 10 5 CFU/mL for bacteria and 5 × 10 4 CFU/mL for yeasts.Proper positive (inoculum without tested compound) and negative (compound without inoculum) controls were added in each microplate.After incubation (35 • C, 24 h), the growth of microorganisms was measured spectrophotometrically at 600 nm (BioTEK ELx808, Bio-Tek Instruments, Inc., Winooski, VT, USA).MICs were marked at the lowest concentration of the compound without the growth of bacteria or fungi.

DPPH Assay
The DPPH radical scavenging assay of samples was performed with ascorbic acid as a positive control [88].Briefly, 100 µL of different concentrations of the complexes, dissolved in DMSO, was mixed with 100 µL of 0.06 mM DPPH methanolic solution and incubated at room temperature in the dark for 30 min.The change in absorbance at λ = 517 nm was analyzed with the use of a 96-well microplate reader (Epoch, BioTek System, Winooski, VT, USA).The control was composed of DPPH and DMSO.
DPPH inhibition was calculated according to the following equation: DPPH Inhibition (%) = [(Acontrol − Asample)/Acontrol] × 100% The radical scavenging activity of the samples was shown as the IC 50 value (the concentration of the analyzed samples that caused a decrease in the non-reduced form of the DPPH radical by 50%).

ABTS Assay
The ABTS radical scavenging assay of samples (ligands or complexes) was performed with ascorbic acid as a positive control [88].Briefly, 30 µL of different concentrations of the samples, dissolved in DMSO, was mixed with 170 µL of ABTS solution (2 mM ABTS diammonium salt, 3.5 mM potassium persulfate) and completed with water to a final volume of 300 µL.ABTS solution with DMSO was used as a control.After 10 min of incubation at 30 • C in the dark, a change in absorbance was observed at λ = 750 nm by a 96-well microplate reader (Epoch, BioTek System, Santa Clara, CA, USA).
ABTS inhibition was calculated according to the following equation: ABTS Inhibition (%) = [(Acontrol − Asample)/Acontrol] × 100% The radical scavenging activity of the samples was shown as the IC 50 value (the concentration of the analyzed samples that caused a decrease in the non-reduced form of the ABTS radical by 50%).

ADME/Drug-Likeness Calculation
The physicochemical, pharmacokinetic and drug-likeness properties of copper(II) complexes C1-4 were predicted using the SwissADME web tool, which is available online [103].

Conclusions
Four new copper(II) complexes, C1-4, derived from 1-(isoquinolin-3-yl)heteroalkyl-2one ligands, L1-4, were prepared, and their coordination modes were established using an elemental analysis, infrared spectra as well as X-ray crystallographic study for C1.The structures of copper(II) complexes C1 and C2 were determined as mononuclear species incorporating two molecules of the neutral 1-(isoquinolin-3-yl)heteroalkyl-2-one ligand bound to the central copper ion via a bidentate manner.Interestingly, ligands L3 and L4 containing an alkyl group at the N-3 position of the imidazolidin-2-one moiety form tetracoordinate mononuclear copper(II) complexes C3 and C4 with the central atom chelated by one molecule of the neutral bidentate ligand.
The results of UV-vis spectrophotometry confirmed the stability of complexes C1-4 under conditions that mimic the physiological environment.
The in vitro cytotoxicity studies showed that the coordination of 1-(isoquinolin-3yl)heteroalkyl-2-one ligands L1-4 with a copper(II) ion results in metal complexes C1-4 with remarkable growth inhibitory properties against tested human cancer cell lines, especially hepatoma HepG2, colon cancer LS-180 and glioblastoma T98G cells.The cytotoxic effect of the synthesized copper(II) complexes towards HepG2, LS-180 and T98G cancer cells was higher than the known antitumor agent etoposide.Among these compounds, dichloro{bis[1-(isoquinolin-3-yl)imidazolidin-2-one]}copper(II) (C2) was found to be the most promising agent with the greatest selective effect on HepG2, LS180 and T98G cancer cell lines compared with the non-cancerous CCD-1059-Sk cell line.The complex C2 induced sub-G1 cell cycle arrest in the HepG2 cells and induced G1/M cell cycle arrest in the T98G cells, which was accompanied by DNA degradation.Furthermore, the treatment of HepG2 and T98G cells with the tested copper(II) compound C2 at the concentration that did not inhibit the growth of cancer cells resulted in a significant increase in the cytotoxic effects of chemotherapeutics such as etoposide, 5-fluorouracil and temozolomide.To clarify the mechanism of synergism between C2 and the clinically used drugs, more advanced studies are needed.
The antioxidant activity results suggest that in the DPPH test, the coordination of ligands L1-4 with a copper(II) metal center is beneficial for antiradical potency.No direct correlation was found between antiproliferative and antioxidant effects; the exception to this was dichloro[1-ethyl-3-(isoquinolin-3-yl)imidazolidin-2-one]copper(II) (C4), which demonstrated remarkable growth-inhibitory properties against cancer cells and the strongest antioxidant activity in both the DPPH and ABTS assays within the tested compounds.On the other hand, the copper(II) compound C4, which had the highest potency against the tested tumor cell lines, exhibited moderate antiradical properties.
Generally, the prediction of ADME/drug-likeness properties revealed that the tested copper(II) complexes may be considered as drug-likeness molecules.
In summary, the results obtained may be useful as a starting point for the development of novel copper-based antitumor agents.

Scheme 3 .
Scheme 3. Synthesis of copper(II) complex C1 in the reaction of ligand L1 with copper(II) chloride dihydrate in dimethylformamide.

Scheme 3 .Scheme 4 .
Scheme 3. Synthesis of copper(II) complex C1 in the reaction of ligand L1 with copper(II) c dihydrate in dimethylformamide.The coordination compounds C2, C3 and C4 were obtained in acceptable yield analogous reaction conditions and copper(II) salt stoichiometries (Scheme 4).It sho emphasized that the ligand unsubstituted at the nitrogen atom in position 3 of the azolidin-2-one; derivative L2 formed a neutral mononuclear chelate C2 (L2CuCl2), the N 3 -substituted ligands L3 and L4 allowed the preparation of brown-green bid N,O-chelates C3 and C4 with the structure LCuCl2.

Figure 3 .
Figure 3. Structural representation and atom-numbering scheme.Thermal ellipsoids are drawn at the 50% probability level.Coordinated ligands are in trans position to each other.The copper ion is in a distorted octahedral environment with two N atoms and two O atoms from two ligands in the equational plane and two Cl donors in the opposite axial sites (Figure 4).The copper ions reside in the center of the octahedron, in which the bond lengths are Cu1 − N1 = 2.0666 (12)Å, Cu1 − O1 = 2.4210 (11) Å and Cu1 − Cl1 = 2.3051 (4) Å.

Figure 3 .
Figure 3. Structural representation and atom-numbering scheme.Thermal ellipsoids are drawn at the 50% probability level.Coordinated ligands are in trans position to each other.The copper ion is in a distorted octahedral environment with two N atoms and two O atoms from two ligands in the equational plane and two Cl donors in the opposite axial sites (Figure 4).The copper ions reside in the center of the octahedron, in which the bond lengths are Cu1 − N1 = 2.0666 (12)Å, Cu1 − O1 = 2.4210 (11) Å and Cu1 − Cl1 = 2.3051 (4) Å. Int.J. Mol.Sci.2024, 25, x FOR PEER REVIEW 8 of 29

Figure 4 .
Figure 4. Crystal packing of copper(II) complex C1 in the unit cell.

Figure 4 .
Figure 4. Crystal packing of copper(II) complex C1 in the unit cell.There are no strong hydrogen bonds in the C1 structure.There are hydrogen bonds of the C-H. ..Cl(O) type.The C-H. ..O bond stabilizes the coordination fragment (Table2).However, the C-H. ..Cl hydrogen bond stabilizes the packing.

Figure 5 .Figure 5 .
Figure 5. Structures of possible rotamers of ligand L2 obtained by rotation along the C3-N1′ a Figure 5. Structures of possible rotamers of ligand L2 obtained by rotation along the C 3 -N 1 axis.

Figure 8 .
Figure 8. Structures of two possible rotamers of ligand L2, and corresponding electronic en

Figure 10 .
Figure 10.Cell cycle analysis of HepG2 (A) and T98G cells (B) incubated for 24 h with copper(II) complex C2 (CX) at its IC50 concentration.Results are expressed as means ± SEM.Statistical significance was designated as **** when p < 0.0001 (vs.control cells) using ANOVA analysis followed by Tukey's post hoc test.

Figure 10 .
Figure 10.Cell cycle analysis of HepG2 (A) and T98G cells (B) incubated for 24 h with copper(II) complex C2 (CX) at its IC 50 concentration.Results are expressed as means ± SEM.Statistical significance was designated as **** when p < 0.0001 (vs.control cells) using ANOVA analysis followed by Tukey's post hoc test.

Table 1 .
X-ray diffraction data and structure refinement details for copper(II) complex C1.

Table 1 .
X-ray diffraction data and structure refinement details for copper(II) complex C1.

Table 3 .
Cytotoxic activity of the investigated free ligands L1-4, Cu(II) complexes C1-4 and reference compound against human cancer cell lines and non-cancerous cells determined by MTT assay after 24 h incubation.
* Etoposide was used as a positive control; IC 50 -the concentration that inhibits 50% of cell viability.The values shown are mean ± SD from three repetitions in two independent experiments.
* MIC-minimum inhibitory concentration in milligrams per liter.
* NR-the IC 50 value was not reached.